Novel proline-rich membrane protein

ABSTRACT

The present invention provides a human proline-rich membrane protein (PRMP) and polynucleotides which identify and encode PRMP. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding PRMP and a method for producing PRMP. The invention also provides for agonists, antibodies, or antagonists specifically binding PRMP, and their use, in the prevention and treatment of diseases associated with expression of PRMP. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding PRMP for the treatment of diseases associated with the expression of PRMP. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding PRMP.

[0001] This application is a continuation application of U.S.application Ser. No. 09/195,862, filed Nov. 19, 1998, which is adivisional application of U.S. application Ser. No. 08,794,216, filedJan. 30, 1997, now U.S. Pat. No. 5,843,716, issued Dec. 1, 1998, bothentitled NOVEL PROLINE-RICH MEMBRANE PROTEIN, all of which applicationsand patents are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a novel proline-rich membrane protein and to the use of thesesequences in the diagnosis, prevention, and treatment of autoimmune andinflammatory diseases and disorders relating to abnormal cellularproliferation including atherosclerosis and cancer.

BACKGROUND OF THE INVENTION

[0003] For the maintenance of an efficient immune defense system,lymphocytes actively migrate between the various lymphoid andnon-lymphoid tissues of the body by way of the bloodstream in order todetect sites of antigen exposure. This migration involves movementthrough intact vascular endothelium and requires interactions betweenreceptors on lymphocytes and ligands displayed by vascular endothelialcells. Exposure to various pro-inflammatory mediators, such ascytokines, lipopolysaccharide endotoxin (LPS), and tumor necrosis factor(TNF), increases the adhesion of lymphocytes to human umbilical veinendothelial cells (HUVECs) by up-regulating the expression ofintracellular adhesion molecules and vascular cell adhesion molecules(ICAMs and VCAMs; Pinola, M. et al. (1992) Scand. J. Immunol.36:671-679). This up-regulation represents an activation state whichprovides characteristic anchorage sites for the increased migration oflymphocytes towards the site of inflammation.

[0004] In the presence of various pro-inflammatory mediators,endothelial cell-derived adhesion molecules initiate the adherence oflymphocytes to endothelium and thus begin the early phases ofimmunologically mediated inflammation. Upon exposure to mediatorspresent at inflamed or infected sites, lymphocytes react with increasedchemotaxis and adherence to endothelium, leading to degranulation,oxidative metabolism, and pathogen killing. Although critical foreffective host defense, these events are also in part responsible fortissue damage associated with inflammation. Abnormalities in lymphocytetrafficking and inflammatory responses are causative factors ininflammatory and autoimmune diseases.

[0005] Adhesion molecules also mediate cell-cell interactions whichcontrol the fate and proliferation of epithelial cells. Interactionsbetween epithelial cells, in the form of specialized junctions, controlcell proliferation, differentiation, and morphogenesis. Apical junctionssuch as adherens junctions are associated with actin microfilaments, andat least one group of cell adhesion molecules, the cadherins. Thecadherins and their associated anchoring molecules, the catenins, havebeen localized at adherens junctions (Geiger, B. et al. (1992) Ann. Rev.Cell Biol. 8:307-332). Loss of expression of cadherins and relatedmolecules is associated with loss of cell proliferation control (Field,J. K. (1992) Eur. J. Cancer (B) 28B:67-76).

[0006] Adherens junctions also participate in cell-cell interactionsthrough their association with a protein tyrosine kinase (PTK)-mediatedsignaling pathway. Tyrosine phosphorylation at adherens junctions ispartly a function of non-receptor PTKs. Two such kinases, c-Yes andc-Src, are highly enriched in the adherens junctions of hepatocytes,kidney epithelial cells and keratinocytes (Tsukita, S. et al. (1991) J.Cell Biol. 113:876-879). Expression of the oncogenic v-Src in epithelialcells causes abnormally high levels of tyrosine phosphorylation,breakdown of adherens junctions, and loss of cell-cell adhesion(Volberg, T. et al. (1991) Cell Regul. 2:105-120). The cadherin/catenincomplexes are particularly susceptible to oncogenic phosphorylation.Fibroblasts and epithelial cells transformed with v-Src expresscadherins which become hyperphosphorylated and are thus unable tofunction properly in cell adhesion or metastasis suppression (Hamaguchi,M. et al. (1993) EMBO J. 12:307-314).

[0007] Many polypeptide hormones, cytokines, antigens, and components ofthe extracellular matrix bind membrane-spanning receptors which signalthrough associated cytoplasmic non-receptor PTK domains. Although thetargets of these PTKs may have vastly different biochemical activitiesand biological functions, they often recognize related sequenceelements. These sequence elements, known as Src-homology-2 (SH2),Src-homology-3 (SH3), and pleckstrin homology (PH) domains, can foldinto independent, compact binding modules. SH2 domains bind shortphosphotyrosine-containing peptide motifs, SH3 domains bind shortpeptide motifs which contain one or more proline residues, and PHdomains may associate with phospholipids. These conserved proteindomains form “binding modules” (Pawson, T. (1995) Nature 373:573-580)which mediate intermolecular protein-protein associations.

[0008] The SH3 binding module consists of the complex of a proline-richpeptide domain (PRD) on one protein with an SH3 domain on anotherprotein. The PRD usually consists of seven to ten amino acids andcontains the consensus sequence X-P-X-X-P, where X is usually analiphatic residue. The PRD forms a left-handed polyproline type IIhelix. Each X-P pair fits into a hydrophobic pocket formed by conservedaromatic residues of the SH3 domain (Feng, S. et al. (1994) Science1241-1246; Lim, W. A. et al. (1994) Nature 372:375-379).

[0009] The conserved noncatalytic domains of many non-receptor PTKs arerequired for intermolecular interactions with activators and effectors,as well as intramolecular regulatory interactions (Bunnell S. C. et al.(1996) J. Biol. Chem. 271:25646-25656; Pawson, T. et al. (1992) Cell71:359-362). PRDs in several such kinases, such as Btk and Itk, arerecognized by the SH3 domains of various Src family kinases. The PRDs inBtk and Itk contain the consensus sequence XPΦPPXP, where Φ denotes ahydrophobic residue (Yang, W. et al. (1995) J. Biol. Chem270:20832-20840).

[0010] Mutations in Btk have been associated with immunodeficiencies inman and mouse. In addition, Btk is found in association with an as yetunidentified 72-kDa phosphotyrosine-containing protein; this interactionrequires a functional PRD in Btk (Yang et al., supra). The SH3-bindingPRD of Btk may therefore interact in vivo with proteins that regulatethe phosphorylation state of Btk and thus regulate the participation ofBtk in various receptor-mediated signaling pathways (Yang et al.,supra).

[0011] Numerous mammalian ion channels such as the human Kv1.5 potassiumchannel (hKv1.5) contains PRDs. Direct association of the SH3 domain ofSrc tyrosine kinase with the PRDs of hKv1.5 was observed (Holmes, T. C.et al. (1996) Science 274:2089-2091). Holmes et. al. propose thatclosely associated channel-kinase signaling complexes may serve toincrease the specificity of signaling pathways.

[0012] Subunits of the N-methyl D-aspartate (NMDA) receptor complexcontain PRDs which may interact with SH3 domain-containing signalingmolecules. The NMDA receptor complex is a postsynaptic cation channelactivated by the excitatory neurotransmitter glutamic acid and specificfor the agonist NMDA. A putative NMDA receptor glutamate-binding proteinwhich contains PRDs in the N-terminal region has been cloned from ratbrain (Kumar, K. N. et al. (1991) Nature 354:70-73). While this proteinexhibits the binding characteristics of an NMDA receptor subunit (Kumar,supra), the role of the glutamate-binding protein as an NMDA receptorsubunit has been questioned (Nakanishi, S. (1992) Science 258:597-603).

[0013] Proteins which contain SH3 domains or SH3-binding PRDs are alsoimportant for cellular organization and the control of cellularmorphology. Several proteins associated with the cytoskeleton, includingα-spectrin and myosin-1, contain SH3 domains (Pawson 1995, supra).Numerous SH3 domain-containing proteins in yeast are required fororganization or polarization of the actin cytoskeleton (Pawson 1995,supra).

[0014] Mutations in the SH3 domain-containing Drosophilatumor-suppressor gene discs large (dlg) lead to a loss of the tight(septate) junctions between epithelial cells and result in loss ofapical-basal polarity and aberrant cell proliferation (Woods, D. F. etal. (1993) J. Cell Sci, Suppl. 17:171-181). Dlg protein is expressed inmost epithelial tissues throughout development. Potential ligands forthe mammalian homologues of Dlg include the small GTP-binding proteinrho. Rho binds with high affinity to SH3 domains, is involved in actinbundling, and regulates the assembly of focal adhesions (Ridley, A. J.et al. (1992) Cell 70:389-399; Woods et al., supra).

[0015] In phagocytes, the NADPH oxidase multiprotein complex isactivated by inflammatory stimuli to produce superoxide, a precursor forantimicrobial oxidants. This activation is dependent on the interactionof SH3 domain-containing oxidase proteins p47-phox, p67-phox, andp40-phox with other proteins of the oxidase complex (McPhail, L. C.(1994) J. Exp. Med. 180:2011-2015). The SH3 domains of p47-phox andp67-phox may be responsible for assembly of the functional oxidasecomplex (Pawson 1995, supra). For instance, a mutation of proline toglutamine in the PRD of the oxidase component p22-phox was detected in apatient with chronic granulomatous disease, a condition characterized byhigh susceptibility to bacterial and fungal infections. The mutationblocked the interaction of p22-phox with the p47-phox SH3 domain.

[0016] The discovery of polynucleotides encoding a novel proline-richmembrane protein, and the molecules themselves, provides a means toinvestigate cell signaling, protein trafficking and subcellularlocalization, the control of cellular architecture, cell-cellinteractions, cellular proliferation, and inflammatory and immuneresponses under normal and disease conditions. Discovery of a novelproline-rich membrane protein satisfies a need in the art by providingnew compositions useful in diagnosing and treating autoimmune andinflammatory diseases and disorders relating to abnormal cellularproliferation including atherosclerosis and cancer.

SUMMARY OF THE INVENTION

[0017] The present invention features a novel proline-rich membraneprotein hereinafter designated PRMP and characterized as havingsimilarity to rat NMDA receptor glutamic acid binding subunit.

[0018] Accordingly, the invention features a substantially purified PRMPhaving the amino acid sequence shown in SEQ ID NO: 1.

[0019] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode PRMP. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0020] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0021] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode PRMP. Thepresent invention also features antibodies which bind specifically toPRMP, and pharmaceutical compositions comprising substantially purifiedPRMP. The invention also features the use of agonists and antagonists ofPRMP. The invention also features methods for treating disorders whichare associated with PRMP, and for detecting a polynucleotide whichencodes PRMP.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ IDNO:1) and nucleic acid sequence (SEQ ID NO:2) of PRMP. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd., San Bruno, Calif.).

[0023]FIGS. 2A and 2B show the amino acid sequence alignments betweenPRMP (SEQ ID NO:1) and rat NMDA receptor glutamate-binding subunit (GI238267; SEQ ID NO:3). The alignment was produced using the multisequencealignment program of DNASTAR software (DNASTAR Inc, Madison Wis.).

[0024]FIGS. 3A and 3B show the hydrophobicity plots (produced using theprotein analysis program of DNASTAR software) for PRMP, SEQ ID NO: 1,and rat NMDA receptor glutamate-binding subunit, SEQ ID NO:3. Thepositive X axis reflects amino acid position, and the negative Y axis,hydrophobicity.

[0025]FIGS. 4A and 4B show the northern analysis for SEQ ID NO:2. Thenorthern analysis was produced electronically using LIFESEQ database(Incyte Pharmaceuticals, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0026] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0027] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0028] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0029] Definitions

[0030] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0031] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0032] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0033] PRMP, as used herein, refers to the amino acid sequences ofsubstantially purified PRMP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0034] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using an XL-PCR kit (Perkin Elmer, Norwalk, Conn.) in the 5′and/or the 3′ direction and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte clone using theGELVIEW fragment assembly system (GCG, Madison, Wis.), or which has beenboth extended and assembled.

[0035] A “variant” of PRMP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0036] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0037] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0038] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0039] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic PRMP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0040] The term “agonist”, as used herein, refers to a molecule which,when bound to PRMP, causes a change in PRMP which modulates the activityof PRMP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to PRMP.

[0041] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to PRMP, blocks or modulates the biologicalor immunological activity of PRMP. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to PRMP.

[0042] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of PRMP. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of PRMP.

[0043] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of PRMPor portions thereof and, as such, is able to effect some or all of theactions of PRD-containing molecules.

[0044] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding PRMP or the encoded PRMP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0045] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0046] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0047] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0048] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0049] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0050] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0051] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0052] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0053] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0054] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human PRMP and fragments thereof.

[0055] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0056] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0057] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0058] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding PRMPor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0059] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding PRMP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0060] “Alterations” in the polynucleotide of SEQ ID NO: 2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding PRMP including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes PRMP (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO:2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding PRMP (e.g., using fluorescent in situhybridization (FISH) to metaphase chromosomes spreads).

[0061] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fab, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind PRMPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0062] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0063] The Invention

[0064] The invention is based on the discovery of a novel humanproline-rich membrane protein (PRMP), the polynucleotides encoding PRMP,and the use of these compositions for the diagnosis, prevention, ortreatment of autoimmune and inflammatory diseases and disorders relatingto abnormal cellular proliferation including atherosclerosis and cancer.

[0065] Nucleic acids encoding the human PRMP of the present inventionwere first identified in Incyte Clone 155397 from the PMA/LPS-treatedpromonocyte cell line cDNA library (THP1PLB02) through acomputer-generated search for amino acid sequence alignments. Aconsensus sequence, SEQ ID NO:2, was derived from extension of IncyteClone 155397 (THP1PLB02).

[0066] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C, and 1D. PRMP is 311 amino acids in length and has chemical andstructural homology with the central portion of rat NMDA receptorglutamate-binding subunit (GI 238267; SEQ ID NO:3). In particular, PRMPshares 41% identity to the central portion of rat NMDA receptorglutamate-binding subunit (FIGS. 2A and 2B). PRMP contains up to sevenpotential transmembrane domains located approximately at amino acidresidues 102-123, 133-152, 163-182, 191-212, 222-243, 251-271, and288-307 of SEQ ID NO:1. Of particular note is the high proportion ofproline and tyrosine residues in the N-terminal sequence of PRMP priorto the first transmembrane domain (25% pro and 10% tyr), which suggeststhe presence of SH3-binding PRDs and tyrosine phosphorylation sites. Asillustrated by FIGS. 3A and 3B, PRMP has a similar hydrophobicityprofile to the central portion of rat NMDA receptor glutamate-bindingsubunit. Northern analysis (FIGS. 4A and 4B) shows the abundantexpression of PRMP in the HUVEC endothelial cell line activated bypro-inflammatory mediators. In addition. PRMP is found in numerousepithelial and endothelial tissues and cell lines; cells and tissuesinvolved in immune response and inflammation; and tumor-associatedepithelial tissues.

[0067] The invention also encompasses PRMP variants. A preferred PRMPvariant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the PRMP amino acid sequence (SEQ ID NO: 1). A mostpreferred PRMP variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO:1.

[0068] The invention also encompasses polynucleotides which encode PRMP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of PRMP can be used to generate recombinant molecules whichexpress PRMP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, and 1D.

[0069] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding PRMP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring PRMP, and all such variations are to beconsidered as being specifically disclosed.

[0070] Although nucleotide sequences which encode PRMP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PRMP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PRMP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PRMP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0071] The invention also encompasses production of DNA sequences, orportions thereof, which encode PRMP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding PRMP or any portionthereof.

[0072] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0073] Altered nucleic acid sequences encoding PRMP which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent PRMP. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent PRMP. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of PRMP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0074] Also included within the scope of the present invention arealleles of the genes encoding PRMP. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0075] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE polymerase (US Biochemical Corp, Cleveland,Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase(Amersham, Chicago, Ill.), or combinations of recombinant polymerasesand proofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton MICROLAB 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0076] The nucleic acid sequences encoding PRMP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0077] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0078] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0079] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0080] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0081] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0082] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode PRMP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of PRMP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressPRMP.

[0083] As will be understood by those of skill in the art, it may beadvantageous to produce PRMP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0084] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterPRMP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0085] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PRMP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of PRMP activity, it may be useful toencode a chimeric PRMP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the PRMP encoding sequence and theheterologous protein sequence, so that PRMP may be cleaved and purifiedaway from the heterologous moiety.

[0086] In another embodiment, sequences encoding PRMP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of PRMP, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0087] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of PRMP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0088] In order to express a biologically active PRMP, the nucleotidesequences encoding PRMP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0089] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding PRMPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0090] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding PRMP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0091] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding PRMP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0092] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for PRMP. For example, whenlarge quantities of PRMP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding PRMP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of 13-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0093] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0094] In cases where plant expression vectors are used, the expressionof sequences encoding PRMP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0095] An insect system may also be used to express PRMP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding PRMPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of PRMP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which PRMP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0096] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding PRMP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing PRMP in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0097] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding PRMP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding PRMP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0098] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0099] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress PRMP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0100] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0101] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding PRMP isinserted within a marker gene sequence, recombinant cells containingsequences encoding PRMP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding PRMP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0102] Alternatively, host cells which contain the nucleic acid sequenceencoding PRMP and express PRMP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0103] The presence of polynucleotide sequences encoding PRMP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding PRMP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding PRMP todetect transformants containing DNA or RNA encoding PRMP. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0104] A variety of protocols for detecting and measuring the expressionof PRMP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson PRMP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0105] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding PRMPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding PRMP, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0106] Host cells transformed with nucleotide sequences encoding PRMPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode PRMP may be designed to contain signal sequences which directsecretion of PRMP through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingPRMP to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and PRMP may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining PRMP and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying PRMP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll. D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0107] In addition to recombinant production, fragments of PRMP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 43 1APeptide Synthesizer (Perkin Elmer). Various fragments of PRMP may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0108] Therapeutics

[0109] Chemical and structural homology exists between PRMP and NMDAreceptor glutamate-binding subunit from rat. In addition, the presenceof proline-rich domains, and the expression of PRMP ininflammation-activated endothelial cells, tumor-associated tissues ofepithelial origin, and other tissues involved in immune or inflammatorydisorders, suggests that PRMP has a role in cell signaling, proteintrafficking and subcellular localization, control of cell architecture,cell-cell interactions, cell growth and development, and modulation ofimmune and inflammatory responses.

[0110] Therefore, in one embodiment, PRMP or a fragment or derivativethereof may be administered to a subject or cells removed from a subjectto promote tissue or organ regeneration. This embodiment would be ofparticular benefit in promoting regeneration of endothelial orepithelial tissues.

[0111] In another embodiment, a vector capable of expressing PRMP, or afragment or derivative thereof, may also be administered to a subject orcells isolated from a subject to promote tissue or organ regeneration.

[0112] In another embodiment, a vector expressing antisense of thepolynucleotide encoding PRMP may be administered to a subject to treator prevent a disorder which is associated with expression of PRMP. Suchdisorders may include, but are not limited to, inflammatory and allergicconditions such as rheumatoid and osteoarthritis, asthma, allergicrhinitis, atopic dermatitis; autoimmune conditions such as Sjogren'ssyndrome, scleroderma, hyperthyroidism (Grave's disease), systemiclupus, myasthenia gravis, autoimmune thyroiditis, diabetes mellitus,pancreatitis, ulcerative colitis, Crohn's disease, atrophic gastritis,and graft-vs-host disease; disorders relating to abnormal cellulardifferentiation, proliferation, or degeneration, includingarteriosclerosis, atherosclerosis, hyperaldosteronism, hypocortisolism(Addison's disease), hypothyroidism, colorectal polyps, gastric andduodenal ulcers, cancers of hematopoietic cells and lymphoid tissuesincluding leukemias, lymphomas (including Hodgkin's disease),lymphosarcomas and myelomas, and carcinomas of glands, tissues, andorgans involved in secretion or absorption, including prostate,pancreas, lung, tongue, brain, breast, and bladder, adrenal gland,thyroid, liver, uterus, kidney, testes, and organs of thegastrointestinal tract including small intestine, colon, rectum, andstomach.

[0113] In another embodiment, antagonists or inhibitors of PRMP may beadministered to a subject to treat or prevent any of the disordersassociated with expression of PRMP including those listed above. In aparticular aspect, antibodies which are specific for PRMP may be useddirectly as an antagonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress PRMP.

[0114] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0115] Antagonists or inhibitors of PRMP may be produced using methodswhich are generally known in the art. In particular, purified PRMP maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind PRMP.

[0116] Antibodies specific for PRMP may be generated using methods thatare well known in the art. Such antibodies may include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments, and fragments produced by a Fab expression library.Neutralizing antibodies, (i.e., those which inhibit dimer formation) areespecially preferred for therapeutic use.

[0117] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith PRMP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corvnebacterium parvum are especially preferable.

[0118] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to PRMP have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of PRMP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0119] Monoclonal antibodies to PRMP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0120] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producePRMP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:11120-11123).

[0121] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0122] Antibody fragments which contain specific binding sites for PRMPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0123] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between PRMP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PRMP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0124] In another embodiment of the invention, the polynucleotidesencoding PRMP, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding PRMP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding PRMP. Thus, antisense molecules may be used to modulate PRMPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments can be designed from various locations along the coding orcontrol regions of sequences encoding PRMP.

[0125] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingPRMP. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0126] Genes encoding PRMP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes PRMP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0127] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding PRMP, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0128] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding PRMP.

[0129] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0130] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding PRMP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0131] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0132] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0133] Any of the therapeutic methods described above may be applied toany subject n need of such therapy, including, for example, mammals suchas dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0134] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of PRMP,antibodies to PRMP, mimetics, agonists, antagonists, or inhibitors ofPRMP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0135] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0136] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0137] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0138] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0139] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0140] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0141] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0142] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0143] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0144] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0145] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of PRMP, such labeling wouldinclude amount, frequency, and method of administration.

[0146] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0147] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0148] A therapeutically effective dose refers to that amount of activeingredient, for example PRMP or fragments thereof, antibodies of PRMP,agonists, antagonists or inhibitors of PRMP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0149] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0150] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0151] Diagnostics

[0152] In another embodiment, antibodies which specifically bind PRMPmay be used for the diagnosis of conditions or diseases characterized byexpression of PRMP, or in assays to monitor patients being treated withPRMP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for PRMP includemethods which utilize the antibody and a label to detect PRMP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0153] A variety of protocols including ELISA, RIA, and FACS formeasuring PRMP are known in the art and provide a basis for diagnosingaltered or abnormal levels of PRMP expression. Normal or standard valuesfor PRMP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to PRMP under conditions suitable for complex formation Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of PRMPexpressed in subject samples, control and disease, from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0154] In another embodiment of the invention, the polynucleotidesencoding PRMP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPRMP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of PRMP,and to monitor regulation of PRMP levels during therapeuticintervention.

[0155] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PRMP or closely related molecules, may be used to identifynucleic acid sequences which encode PRMP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding PRMP, alleles, or related sequences.

[0156] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the PRMP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring PRMP.

[0157] Means for producing specific hybridization probes for DNAsencoding PRMP include the cloning of nucleic acid sequences encodingPRMP or PRMP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0158] Polynucleotide sequences encoding PRMP may be used for thediagnosis of disorders which are associated with expression of PRMP.Examples of such disorders include inflammatory and allergic conditionssuch as rheumatoid and osteoarthritis, asthma, allergic rhinitis, atopicdermatitis; autoimmune conditions such as Sjögren's syndrome,scleroderma, hyperthyroidism (Grave's disease), systemic lupus,myasthenia gravis, autoimmune thyroiditis, diabetes mellitus,pancreatitis, ulcerative colitis, Crohn's disease, atrophic gastritis,and graft-vs-host disease; disorders relating to abnormal cellulardifferentiation, proliferation, or degeneration, includingarteriosclerosis, atherosclerosis, hyperaldosteronism, hypocortisolism(Addison's disease), hypothyroidism, colorectal polyps, gastric andduodenal ulcers, cancers of hematopoietic cells and lymphoid tissuesincluding leukemias, lymphomas (including Hodgkin's disease),lymphosarcomas and myelomas, and carcinomas of glands, tissues, andorgans involved in secretion or absorption, including prostate,pancreas, lung, tongue, brain, breast, and bladder, adrenal gland,thyroid, liver, uterus, kidney, testes, and organs of thegastrointestinal tract including small intestine, colon, rectum, andstomach. The polynucleotide sequences encoding PRMP may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered PRMP expression. Such qualitative or quantitative methods arewell known in the art.

[0159] In a particular aspect, the nucleotide sequences encoding PRMPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding PRMP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding PRMP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0160] In order to provide a basis for the diagnosis of diseaseassociated with expression of PRMP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes PRMP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0161] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0162] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0163] Additional diagnostic uses for oligonucleotides designed from thesequences encoding PRMP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′→3′) and another with antisense(3′←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0164] Methods which may also be used to quantitate the expression ofPRMP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0165] In another embodiment of the invention, the nucleic acidsequences which encode PRMP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0166] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding PRMP on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0167] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0168] In another embodiment of the invention, PRMP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenPRMP and the agent being tested, may be measured.

[0169] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to PRMP, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with PRMP, or fragments thereof, and washed. BoundPRMP is then detected by methods well known in the art. Purified PRMPcan also be coated directly onto plates for use in the aforementioneddrug screening techniques. Alternatively, non-neutralizing antibodiescan be used to capture the peptide and immobilize it on a solid support.

[0170] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding PRMPspecifically compete with a test compound for binding PRMP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with PRMP.

[0171] In additional embodiments, the nucleotide sequences which encodePRMP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0172] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0173] I THP1PLB02 cDNA Library Construction

[0174] THP-1 is a human leukemic cell line derived from the blood of a1-year-old boy with acute monocytic leukemia. Cells used for the PMA+LPSlibrary (THP1PLB02) were cultured for 48 hr with 100 nm PMA in DMSO andfor 4 hr with 1 μg/ml LPS. The PMA+LPS-stimulated cells representactivated macrophages. The cDNA library was custom constructed byStratagene essentially as described below.

[0175] Stratagene prepared the cDNA library using oligo d(T) priming.Synthetic adapter oligonucleotides were ligated onto the cDNA moleculesenabling them to be inserted into the UNIZAP vector system (Stratagene).The PBLUESCRIPT phagemid (Stratagene) was excised and transfected intoXL1-BLUE E. coli host strain (Stratagene).

[0176] II Isolation and Sequencing of cDNA Clones

[0177] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an f1 helper phage. Enzymesderived from both the library-containing phage and the helper phagenicked the DNA, initiated new DNA synthesis from defined sequences onthe target DNA, and created a smaller, single stranded circular phagemidDNA molecule that included all DNA sequences of the PBLUESCRIPT phagemid(Stratagene) and the cDNA insert. The phagemid DNA was released from thecells, purified, and used to reinfect fresh host cells (SOLR,Stratagene) where double-stranded phagemid DNA was produced. Because thephagemid carries the gene for β-lactamase, the newly transformedbacteria were selected on medium containing ampicillin.

[0178] Phagemid DNA was released from cells and purified using aMINIPREP kit (Cat. No. 77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Cat. No. 2271 1, LIFE TECHNOLOGIES,Gaithersburg Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2)the bacteria were cultured for 24 hours after the wells were inoculatedand then lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R at 2900 rpm for 5 min was performed beforethe contents of the block were added to the primary filter plate; and 4)the optional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

[0179] Alternative methods of purifying phagemid DNA include the use ofa MAGIC MINIPREPS DNA purification system (Cat. No. A7100, Promega) orQIAWELL-8 plasmid, QIAWELL PLUS DNA, and QIAWELL ULTRA DNA purificationsystems (Qiagen, Inc.).

[0180] The cDNAs were sequenced by the method of Sanger F. and A. R.Coulson (1975; J. Mol. Biol. 94:441f), using a Catalyst 800 (PerkinElmer) or Hamilton Micro Lab 2200 (Hamilton, Reno Nev.) in combinationwith four Peltier Thermal Cyclers (PTC200 from MJ Research, WatertownMass.) and Applied Biosystems 377 or 373 DNA Sequencing Systems (PerkinElmer) and reading frame was determined.

[0181] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0182] Each cDNA was compared to sequences in the GenBank and EMBLdatabases using two homology search algorithms. The first algorithm wasoriginally developed by Lipman D. J. and Pearson W. R. (1985; Science227:1435). In this algorithm, the homologous regions are searched in atwo-step manner. In the first step, highly homologous regions aredetermined by calculating a matching score using a homology score table.In this step, the parameter “Ktup” is used to establish a shifting,minimum window size for comparing two sequences. Ktup also sets thenumber of bases that must match to extract the highest homologous regionamong the sequences. In this step, no insertions or deletions areapplied, and the homology is displayed as an initial (INIT) value.

[0183] In the second step, the homologous regions are aligned to obtainthe highest matching score by inserting a gap when it is needed toaccommodate a probable deletion. The matching score obtained in thefirst step is recalculated using the homology score table and theinsertion score table to produce an optimized value.

[0184] DNA homologies between two sequences may also be examinedgraphically using the Harr method of constructing dot matrix homologyplots (Needleman, S. B. and Wunsch, C. O. (1970) J. Mol. Biol. 48:443).This method produces a two-dimensional plot which can be useful indistinguishing between regions of homology and regions of repetition.

[0185] The second algorithm was developed by Applied Biosystems andincorporated into the INHERIT 670 sequence analysis system. In thisalgorithm, Pattern Specification Language (TRW Inc, Los Angeles, Calif.)was used to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search.

[0186] Peptide and protein sequence homologies were ascertained usingthe INHERIT-670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0187] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al.(1990) J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0188] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0189] IV Northern Analysis

[0190] Northern analysis, a laboratory technique used to detect thepresence of a transcript of a gene, involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0191] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0192] The basis of the search is the product score which is defined as:$\frac{\% \quad \text{sequence identity} \times \% \quad \text{maximum}\quad {BLAST}\quad \text{score}}{100}$

[0193] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0194] The results of northern analysis are reported as a list oflibraries in which the transcript encoding PRMP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0195] V Extension of PRMP-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0196] Full length PRMP-encoding nucleic acid sequence (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0197] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0198] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200: M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0199] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0200] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C, the whole transformation mixtureis plated on Luria Bertani (LB)-agar (Sambrook et al., supra) containing2× Carb. The following day, several colonies are randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× Carb medium placed inan individual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture is transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample is transferred into a PCRarray.

[0201] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0202] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0203] VI Labeling and Use of Hybridization Probes

[0204] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with a SEPHADEX G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN).

[0205] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a PhosphorImager cassette, (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0206] VII Antisense Molecules

[0207] Antisense molecules to the PRMP-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring PRMP. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of PRMP, as shown in FIGS. 1A, 1B, 1C, and 1D,is used to inhibit expression of naturally occurring PRMP. Thecomplementary oligonucleotide is designed from the most unique 5′sequence as shown in FIGS. 1A, 1B, 1C, and 1D and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an PRMP-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5′ coding sequence of the polypeptide asshown in FIGS. 1A, 1B, 1C, and 1D.

[0208] VIII Expression of PRMP

[0209] Expression of PRMP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express PRMP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0210] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of PRMP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0211] IX Demonstration of PRMP Activity

[0212] To assay the ability of PRMP to bind to SH3 domains in vitro, abatch adsorption method is used. DNA encoding the SH3 domain of Srckinase (or other SH3 domain-containing protein) is cloned into a pGEXvector (Promega) and expressed in E. coli as a glutathione-S-transferase(GST) fusion protein. The SH3 domain-GST fusion protein is affixed toglutathione-SEPHAROSE beads (Pharmacia & Upjohn) to form an SH3 domainaffinity matrix (Yang et. al, supra). PRMP is incubated with theaffinity matrix with gentle rocking for 1 hour at 4° C. The matrix isthen washed three times with 20 mM tris-Cl pH 8.3, 150 mM NaCl, 0.5%Nonidet P-40. Bound proteins are eluted by boiling in SDS sample bufferfor 5 min and are fractionated on 7.5% or 10% SDS-PAGE (Sambrook,supra). PRMP is identified by immunoblotting with PRMP specificantibodies.

[0213] X Production of PRMP Specific Antibodies

[0214] PRMP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0215] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0216] XI Purification of Naturally Occurring PRMP Using SpecificAntibodies

[0217] Naturally occurring or recombinant PRMP is substantially purifiedby immunoaffinity chromatography using antibodies specific for PRMP. Animmunoaffinity column is constructed by covalently coupling PRMPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0218] Media containing PRMP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of PRMP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/PRMP binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and PRMP is collected.

[0219] XII Identification of Molecules which Interact with PRMP

[0220] PRMP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled PRMP, washed and any wells withlabeled PRMP complex are assayed. Data obtained using differentconcentrations of PRMP are used to calculate values for the number,affinity, and association of PRMP with the candidate molecules.

[0221] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 3 311 amino acids amino acid single linear Consensus Consensus 1 MetSer Asn Pro Ser Ala Pro Pro Pro Tyr Glu Asp Arg Asn Pro Leu 1 5 10 15Tyr Pro Gly Pro Pro Pro Pro Gly Gly Tyr Gly Gln Pro Ser Val Leu 20 25 30Pro Gly Gly Tyr Pro Ala Tyr Pro Gly Tyr Pro Gln Pro Gly Tyr Gly 35 40 45His Pro Ala Gly Tyr Pro Gln Pro Met Pro Pro Thr His Pro Met Pro 50 55 60Met Asn Tyr Gly Pro Gly His Gly Tyr Asp Gly Glu Glu Arg Ala Val 65 70 7580 Ser Asp Ser Phe Gly Pro Gly Glu Trp Asp Asp Arg Lys Val Arg His 85 9095 Thr Phe Ile Arg Lys Val Tyr Ser Ile Ile Ser Val Gln Leu Leu Ile 100105 110 Thr Val Ala Ile Ile Ala Ile Phe Thr Phe Val Glu Pro Val Ser Ala115 120 125 Phe Val Arg Arg Asn Val Ala Val Tyr Tyr Val Ser Tyr Ala ValPhe 130 135 140 Val Val Thr Tyr Leu Ile Leu Ala Cys Cys Gln Gly Pro ArgArg Arg 145 150 155 160 Phe Pro Trp Asn Ile Ile Leu Leu Thr Leu Phe ThrPhe Ala Met Gly 165 170 175 Phe Met Thr Gly Thr Ile Ser Ser Met Tyr GlnThr Lys Ala Val Ile 180 185 190 Ile Ala Met Ile Ile Thr Ala Val Val SerIle Ser Val Thr Ile Phe 195 200 205 Cys Phe Gln Thr Lys Val Asp Phe ThrSer Cys Thr Gly Leu Phe Cys 210 215 220 Val Leu Gly Ile Val Leu Leu ValThr Gly Ile Val Thr Ser Ile Val 225 230 235 240 Leu Tyr Phe Gln Tyr ValTyr Trp Leu His Met Leu Tyr Ala Ala Leu 245 250 255 Gly Ala Ile Cys PheThr Leu Phe Leu Ala Tyr Asp Thr Gln Leu Val 260 265 270 Leu Gly Asn ArgLys His Thr Ile Ser Pro Glu Asp Tyr Ile Thr Gly 275 280 285 Ala Leu GlnIle Tyr Thr Asp Ile Ile Tyr Ile Phe Thr Phe Val Leu 290 295 300 Gln LeuMet Gly Asp Arg Asn 305 310 2437 base pairs nucleic acid single linearConsensus Consensus 2 ATGCCAGCCC CAAACCTCAT CCCTAGTGGA GGCCTTGCTGATGTGGAAGT GGCCAGGGCC 60 CTCATGGTAG GCTGGGCAGA AGCCCAAGAA CAGGCTCTAAAGCTGCTAAA CCCGGCAGTC 120 CTGGTCCCCG GAGGCTCTTG CCAGTCTGAC AGTGTTCTTGGCACTGCTCA AAGGTCCCAG 180 CAGCTGGGGT TCCCCGTCAG CCCGTGAGCG GCCATGTCCAACCCCAGCGC CCCACCACCA 240 TATGAAGACC GCAACCCCCT GTACCCAGGC CCTCCGCCCCCTGGGGGCTA TGGGCAGCCA 300 TCTGTCCTGC CAGGAGGGTA TCCTGCCTAC CCTGGCTACCCGCAGCCTGG CTACGGTCAC 360 CCTGCTGGCT ACCCACAGCC CATGCCCCCC ACCCACCCGATGCCCATGAA CTACGGCCCA 420 GGCCATGGCT ATGATGGGGA GGAGAGAGCG GTGAGTGATAGCTTCGGGCC TGGAGAGTGG 480 GATGACCGGA AAGTGCGACA CACTTTTATC CGAAAGGTTTACTCCATCAT CTCCGTGCAG 540 CTGCTCATCA CTGTGGCCAT CATTGCTATC TTCACCTTTGTGGAACCTGT CAGCGCCTTT 600 GTGAGGAGAA ATGTGGCTGT CTACTACGTG TCCTATGCTGTCTTCGTTGT CACCTACCTG 660 ATCCTTGCCT GCTGCCAGGG ACCCAGACGC CGTTTCCCATGGAACATCAT TCTGCTGACC 720 CTTTTTACTT TTGCCATGGG CTTCATGACG GGCACCATTTCCAGTATGTA CCAAACCAAA 780 GCCGTCATCA TTGCAATGAT CATCACTGCG GTGGTATCCATTTCAGTCAC CATCTTCTGC 840 TTTCAGACCA AGGTGGACTT CACCTCGTGC ACAGGCCTCTTCTGTGTCCT GGGAATTGTG 900 CTCCTGGTGA CTGGGATTGT CACTAGCATT GTGCTCTACTTCCAATACGT TTACTGGCTC 960 CACATGCTCT ATGCTGCTCT GGGGGCCATT TGTTTCACCCTGTTCCTGGC TTACGACACA 1020 CAGCTGGTCC TGGGGAACCG GAAGCACACC ATCAGCCCCGAGGACTACAT CACTGGCGCC 1080 CTGCAGATTT ACACAGACAT CATCTACATC TTCACCTTTGTGCTGCAGCT GATGGGGGAT 1140 CGCAATTAAG GAGCAAGCCC CCATTTTCAC CCGATCCTGGGCTCTCCCTT CCAAGCTAGA 1200 GGGCTGGGCC CTATGACTGT GGTCTGGGCT TTAGGCCCCTTTCCTTCCCC TTGAGTAACA 1260 TGCCCAGTTT CCTTTCTGTC CTGGAGACAG GTGGCCTCTCTGGCTATGGA TGTGTGGGTA 1320 CTTGGTGGGG ACGGAGGAGC TAGGGACTAA CTGTTGCTCTTGGTGGGCTT GGCAGGGACT 1380 AGGCTGAAGA TGTGTCTTCT CCCCGCCACC TACTGTATGACACCACATTC TTCCTAACAG 1440 CTGGGGTTGT GAGGAATATG AAAAGAGCCT ATTCGATAGCTAGAAGGGAA TATGAAAGGT 1500 AGAAGTGACT TCAAGGTCAC GAGGTTCCCC TCCCACCTCTGTCACAGGCT TCTTGACTAC 1560 GTAGTTGGAG CTATTTCTTC CCCCAGCAAA GCCAGAGAGCTTTGTCCCCG GCCTCCTGGA 1620 CACATAGGCC ATTATCCTGT ATTCCTTTGG CTTGGCATCTTTTAGCTCAG GAAGGTAGAA 1680 GAGATCTGTG CCCATGGGTC TCCTTGCTTC AATCCCTTCTTGTTTCAGTG ACATATGTAT 1740 TGTTTATCTG GGTTAGGGAT GGGGGACAGA TAATAGAACGAGCAAAGTAA CCTATACAGG 1800 CCAGCATGGA ACAGCATCTC CCCTGGGCTT GCTCCTGGCTTGTGACGCTA TAAGACAGAG 1860 CAGGCCACAT GTGGCCATCT GCTCCCCATT CTTGAAAGCTGCTGGGGCCT CCTTGCAGGC 1920 TTCTGGATCT CTGGTCAGAG TGAACTCTTG CTTCCTGTATTCAGGCAGCT CAGAGCAGAA 1980 AGTAAGGGGC AGAGTCATAC GTGTGGCCAG GAAGTAGCCAGGGTGAAGAG AGACTCGGTG 2040 CGGGCAGGGA GAATGCCTGG GGGTCCCTCA CCTGGCTAGGGAGATACCGA AGCCTACTGT 2100 GGTACTGAAG ACTTCTGGGT TCTTTCCTTC TGCTAACCCAGGGAGGGTCC TAAGAGGAAG 2160 GTGACTTCTC TCTGTTTGTC TTAAGTTGCA CTGGGGGATTTCTGACTTGA GGCCCATCTC 2220 TCCAGCCAGC CACTGCCTTC TTTGTAATAT TAAGTGCCTTGAGCTGGAAT GGGGAAGGGG 2280 GACAAGGGTC AGTCTGTCGG GTGGGGGCAG AAATCAAATCAGCCCAAGGA TATAGTTAGG 2340 ATTAATTACT TAATAGAGAA ATCCTAACTA TATCACACAAAGGGATACAA CTATAAATGT 2400 AATAAAATTT ATGTCTAGAA GTTAAAAAAA AAAAAAA 2437516 amino acids amino acid single linear GenBank 238267 3 Met Lys ArgVal Ser Trp Ser Leu Gly Thr Ala Ile Leu Pro Gln Thr 1 5 10 15 Leu AlaIle Leu Trp Gly His Lys Pro Leu Cys Leu Pro Met Phe Ser 20 25 30 Leu ProThr Leu Gly Pro His Thr His Arg Pro Leu Ser Ser Pro Leu 35 40 45 Pro MetVal Asn Gln Gly Ile Pro Met Val Pro Val Pro Ile Thr Arg 50 55 60 Trp LeuPro Leu Lys Asp Leu Leu Lys Glu Ala Thr His Gln Gly His 65 70 75 80 TyrPro Gln Ser Pro Phe Pro Pro Asn Pro Tyr Gly Gln Pro Pro Pro 85 90 95 PheGln Asp Pro Gly Ser Pro Gln His Gly Asn Tyr Gln Glu Glu Gly 100 105 110Pro Pro Ser Tyr Tyr Asp Asn Gln Asp Phe Pro Ser Val Asn Trp Asp 115 120125 Lys Ser Ile Arg Gln Ala Phe Ile Arg Lys Val Phe Leu Val Leu Thr 130135 140 Leu Gln Leu Ser Val Thr Leu Ser Thr Val Ala Ile Phe Thr Phe Val145 150 155 160 Gly Glu Val Lys Gly Phe Val Arg Ala Asn Val Trp Thr TyrTyr Val 165 170 175 Ser Tyr Ala Ile Phe Phe Ile Ser Leu Ile Val Leu SerCys Cys Gly 180 185 190 Asp Phe Arg Lys Lys His Pro Trp Asn Leu Val AlaLeu Ser Ile Leu 195 200 205 Thr Ile Ser Leu Ser Tyr Met Val Gly Met IleAla Ser Phe Tyr Asn 210 215 220 Thr Glu Ala Val Ile Met Ala Val Gly IleThr Thr Ala Val Cys Phe 225 230 235 240 Thr Val Val Ile Phe Ser Met GlnThr Arg Tyr Asp Phe Thr Ser Cys 245 250 255 Met Gly Val Leu Leu Val SerVal Val Val Leu Phe Ile Phe Ala Ile 260 265 270 Leu Cys Ile Phe Ile ArgAsn Arg Ile Leu Glu Ile Val Tyr Ala Ser 275 280 285 Leu Gly Ala Leu LeuPhe Thr Cys Phe Leu Ala Val Asp Thr Gln Leu 290 295 300 Leu Leu Gly AsnLys Gln Leu Ser Leu Ser Pro Glu Glu Tyr Val Phe 305 310 315 320 Ala AlaLeu Asn Leu Tyr Thr Asp Ile Ile Asn Ile Phe Leu Tyr Ile 325 330 335 LeuThr Ile Ile Gly Arg Ser Gln Gly Ile Gly Gln Ala Pro Ala Gln 340 345 350Val Ala Trp Trp Ala Gln Thr His Ala Pro Gly Met Thr Leu Pro Ser 355 360365 Val Leu Pro Pro Leu Trp Phe Pro Ala Met Ala Trp Ser Arg Gly Ser 370375 380 Pro Ser Arg Pro Arg Val Cys Thr Leu Gln Ile Leu Asn Val Arg Thr385 390 395 400 Leu Ser Ala Thr Ala Trp Lys Pro Leu Ser Leu Leu Pro LeuPro Arg 405 410 415 Gly Asp Arg Ala Ala Phe Leu Cys His Leu Leu Ser ThrHis Cys Cys 420 425 430 Met Ser Pro Val Cys Gln Pro Ile Pro Gly Ser GlyIle Asn Thr Arg 435 440 445 Ser Gln Gly Arg Arg Ile Ile Pro Arg Gly GluGly Ala Arg Leu Pro 450 455 460 Ser Cys Pro Ser Ser Pro Gly Ile Glu SerPro Cys Pro Leu Leu Thr 465 470 475 480 Leu Pro Ser Glu Gly Leu Ala GlyTrp Gly Leu Val Leu Val Leu Gly 485 490 495 Pro Glu Thr Lys Arg Gly TrpHis Val Ser Gly Glu Arg Leu Ser Cys 500 505 510 Val Leu Pro Leu 515

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence of SEQ ID NO:1, b) a naturallyoccurring polypeptide comprising an amino acid sequence at least 90%identical to an amino acid sequence of SEQ ID NO:1, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence of SEQ IDNO:1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1,having a sequence of SEQ ID NO:1.
 3. An isolated polynucleotide encodinga polypeptide of claim
 1. 4. An isolated polynucleotide encoding apolypeptide of claim
 2. 5. An isolated polynucleotide of claim 4, havinga sequence of SEQ ID NO:2.
 6. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. A method ofclaim 9, wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide comprising a sequence selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO:2, b) a naturally occurring polynucleotidecomprising a polynucleotide sequence at least 90% identical to apolynucleotide sequence of SEQ ID NO:2, c) a polynucleotide having asequence complementary to a polynucleotide of a), d) a polynucleotidehaving a sequence complementary to a polynucleotide of b) and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional PRMP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional PRMP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional PRMP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, said method comprising the steps of: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, said method comprising: a) treating a biologicalsample containing nucleic acids with the test compound; b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of PRMP in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of PRMP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofPRMP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11comprising: a) immunizing an animal with a polypeptide having an aminoacid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response; b) isolating antibodies fromsaid animal; and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence of SEQ IDNO:1.
 37. An antibody produced by a method of claim
 36. 38. Acomposition comprising the antibody of claim 37 and a suitable carrier.39. A method of making a monoclonal antibody with the specificity of theantibody of claim 11 comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID NO:1.
 40. A monoclonal antibody produced by a method of claim39.
 41. A composition comprising the antibody of claim 40 and a suitablecarrier.
 42. The antibody of claim 11, wherein the antibody is producedby screening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method for detecting a polypeptide havingan amino acid sequence of SEQ ID NO:1 in a sample, comprising the stepsof: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID NO:1 in the sample.
 45. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A polynucleotide of claim 12, comprising the polynucleotidesequence of SEQ ID NO:2.